The overall objective of the proposed research is to characterize the mechanism of interstitial collagen degradation by cathepsin K, the most potent mammalian collagenase presently known. Previously, cathepsin K was identified as the predominant cysteine protease in osteoclasts and the major type II collagen degrading activity in synovial fibroblasts. Unique among structurally related cathepsins and MMP collagenases, cathepsin K cleaves interstitial collagens at multiple intra-helical sites and similar to bacterial collagenases produces low molecular weight peptide fragments from type I and II collagens. It was demonstrated by the principal investigator that the collagenolytic activity of cathepsin K is dramatically enhanced in the presence of glycosaminoglycans (GAGs) and that the protease forms defined high molecular complexes with chondroitin sulfate, the major GAG in cartilage and bone. The proposed working hypotheses are that the collagenolytic activity of cathepsin K requires a specific complex formation with defined GAGs and that the complex exhibits a helicase as well as cleavage activity towards native triple-helical collagens. Furthermore, it is hypothesized that this complex form is unique for cathepsin K, and that the protease complex is the major intracellular collagen-degrading activity in mammalian cells. The physico-kinetic parameters of complex formation, the substrate specificity of the monomeric and complex forms of cathepsin K, the structural foundation of complex formation and specificity will be determined, and novel strategies to selectively inhibit the collagenolytic activity of the enzyme by inhibiting complex formation will be explored. Furthermore, the essential role of cathepsin K complexes in human collagen turnover will be determined. To achieve these goals, a wide range of biochemical, biophysical, cell-biological and structure-analytical methods will be employed. Altogether, these studies will characterize a novel mechanism for the bulk collagen degradation in mammalian tissues and may offer a new strategy to block excessive collagen degradation in diseases such as osteoporosis and arthritis.